International Journal of Molecular Sciences

Article A Enhances Pro-Opiomelanocortin Transcription Regulated by BMP-4 in Mouse Corticotrope AtT20 Cells

Satoshi Fujisawa 1, Motoshi Komatsubara 1, Naoko Tsukamoto-Yamauchi 1, Nahoko Iwata 2, Takahiro Nada 2, Jun Wada 1 and Fumio Otsuka 2,*

1 Department of Nephrology, Rheumatology, Endocrinology and , Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, 2-5-1 Shikata-cho, Kitaku, Okayama 700-8558, Japan; [email protected] (S.F.); swe_etfi[email protected] (M.K.); [email protected] (N.T.-Y.); [email protected] (J.W.) 2 Department of General Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, 2-5-1 Shikata-cho, Kitaku, Okayama 700-8558, Japan; nao53mayfl[email protected] (N.I.); [email protected] (T.N.) * Correspondence: [email protected]; Tel.: +81-86-235-7342; Fax: +81-86-222-7345

Abstract: Orexin is expressed mainly in the and is known to activate the hypothalamic– pituitary–adrenal (HPA) axis that is involved in various stress responses and its resilience. However, the effects of orexin on the endocrine function of pituitary corticotrope cells remain unclear. In this  study, we investigated the roles of orexin A in pro-opiomelanocortin (POMC) transcription using  mouse corticotrope AtT20 cells, focusing on the bone morphogenetic protein (BMP) system expressed Citation: Fujisawa, S.; Komatsubara, in the pituitary. Regarding the receptors for orexin, type 2 (OXR2) rather than type 1 (OX1R) M.; Tsukamoto-Yamauchi, N.; Iwata, mRNA was predominantly expressed in AtT20 cells. It was found that orexin A treatment enhanced N.; Nada, T.; Wada, J.; Otsuka, F. POMC expression, induced by corticotropin-releasing hormone (CRH) stimulation through upregu- Orexin A Enhances lation of CRH receptor type-1 (CRHR1). Orexin A had no direct effect on the POMC transcription Pro-Opiomelanocortin Transcription suppressed by BMP-4 treatment, whereas it suppressed Smad1/5/9 phosphorylation and Id-1 mRNA Regulated by BMP-4 in Mouse expression induced by BMP-4. It was further revealed that orexin A had no significant effect on the Corticotrope AtT20 Cells. Int. J. Mol. expression levels of type I and II BMP receptors but upregulated inhibitory Smad6/7 mRNA and Sci. 2021, 22, 4553. https://doi.org/ 10.3390/ijms22094553 protein levels in AtT20 cells. The results demonstrated that orexin A upregulated CRHR signaling and downregulated BMP-Smad signaling, leading to an enhancement of POMC transcription by Academic Editors: corticotrope cells. Kazunori Kageyama and Takahiro Nemoto Keywords: anterior pituitary; bone morphogenetic protein (BMP); corticotrope; orexin; pro-opiomelanocortin (POMC) Received: 15 March 2021 Accepted: 25 April 2021 Published: 27 April 2021 1. Introduction Publisher’s Note: MDPI stays neutral are that are expressed primarily in the hypothalamus and with regard to jurisdictional claims in are produced in two isoforms, orexin A (ORX) and orexin B, from a common precursor published maps and institutional affil- protein, prepro-orexin [1]. There are two types of G protein-coupled receptors, named iations. orexin type 1 (OX1R) and type 2 (OX2R) receptors. Orexin B binds only to OX2R, while orexin A binds to both OX1R and OX2R [2]. Since its discovery in 1998, orexin has been reported to have various effects on –wake regulation, feeding behavior, emotions, and the autonomic nervous system involved in stress control [1,3,4]. Orexin is also known to Copyright: © 2021 by the authors. have physiological effects in peripheral tissues, including the endocrine system [5]. Licensee MDPI, Basel, Switzerland. An interaction between the hypothalamic–pituitary–adrenal (HPA) system in response This article is an open access article to stressful stimuli and orexin has been recognized. Orexin has been reported distributed under the terms and to enhance adrenocortical hormone secretion [6,7] in the HPA axis, and orexin neurons conditions of the Creative Commons are further activated by corticotropin-releasing hormone (CRH) [8]. Previous studies Attribution (CC BY) license (https:// showed that an intracerebroventricular injection of orexin increased adrenocorticotropin creativecommons.org/licenses/by/ 4.0/). (ACTH) and corticosterone levels in the blood, which were reversed by CRH receptor

Int. J. Mol. Sci. 2021, 22, 4553. https://doi.org/10.3390/ijms22094553 https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2021, 22, 4553 2 of 10

inhibitors [9–11]. Moreover, that orexin increases CRH and arginine (AVP) mRNA expression in the paraventricular nucleus (PVN) of the hypothalamus [12]. These results suggested that orexin plays an important role in regulating the HPA axis, which is involved in stress responses, mainly in the hypothalamus. Moreover, in the adrenal cortex, it was shown that orexin A rather than orexin B directly stimulates corticosterone secretion in adrenal cortical cells and that this effect is not blunted by ACTH receptor antagonists [13,14]. Those studies showed that orexin has a functional role in the adrenal cortex, independent of ACTH-stimulated control. ACTH is produced from the precursor protein pro-opiomelanocortin (POMC), which is synthesized in corticotrope cells. POMC is also cleaved into multiple hormones in the pituitary and hypothalamus. POMC neurons are located in the arcuate nucleus of the hypothalamus; they suppress , regulate energy balances, and are thought to be involved in the mechanism of orexin-mediated feeding behavior [15]. Increasing evidence has shown a mutual relationship between orexin, the HPA axis, and POMC in the hypothalamus. Given the finding that OX2R was expressed in human corticotrope cells, orexin might be directly involved in the mechanism of hormone production by the anterior pituitary [16]. There has been growing evidence that BMPs, members of the transforming growth factor (TGF)β superfamily of proteins, play functional roles in various endocrine organs in an autocrine or paracrine manner [17]. In pituitary corticotrope AtT20 cells, BMP-4 is known to inhibit the tumorigenesis of corticotrope tumor cells [18,19]. We previously reported that BMP-4 was involved in the suppression of ACTH secretion by receptor stimulation and also suppressed GHRP-2-induced ACTH secretion in AtT20 cells [20,21]. Interestingly, we recently revealed that orexin inhibits secretion by suppressing BMP signaling in rat somatolactotrope GH3 cells [22]. In the present study, we investigated the role of orexin in endocrine regulation in the pituitary gland, focusing on the relationship of orexin with BMP signaling by using the mouse pituitary corticotrope tumor cell line AtT20.

2. Results First, we evaluated the expression of orexin receptors in AtT20 cells. Although the expression of both OX1R and OX2R was detected in mouse pituitary gonadotrope LβT2 cells by RT-PCR, AtT20 cells mainly expressed OX2R, as shown in Figure1A. AtT20 cells express CRHR1 as a receptor for CRH, and orexin A (100 nM) treatment enhanced the mRNA expression of CRHR1. To determine whether this orexin-induced change in the CRH receptor was also detectable at protein levels, Western blotting was performed. As shown in Figure1B, orexin A (100 nM) treatment for 48 h increased the protein expression level of CRHR1 in a time-dependent manner. Therefore, we examined the function of orexin in CRH signaling in AtT-20 cells. Treatment with orexin A (10–300 nM) did not change basal mRNA levels of POMC for 24 h. However, in the presence of CRH (100 nM), co-treatment with orexin (10–300 nM) for 24 h enhanced CRH-induced mRNA expression of POMC (Figure1C). Next, we examined the effect of orexin on the action of BMP-4 in AtT-20 cells. We previously reported that BMP-4 suppressed POMC mRNA expression in AtT20 cells [20]. As shown in Figure2A, BMP-4 (10 ng/mL) treatment for 24 h decreased POMC mRNA, consistent with the results of our previous study. Co-treatment with orexin (10–100 nM) did not change the POMC mRNA expression induced by BMP-4 treatment from 1 h to 24 h (Figure2A). On the other hand, focusing on the effect of orexin on BMP-Smad signaling, stimulation with BMP-4 (10 ng/mL) for 1 h activated Smad1/5/9 phosphorylation in AtT20 cells (Figure2B). Importantly, orexin A (100 nM) pretreatment for 24 h inhibited the phosphorylation of Smad1/5/9 by BMP-4 (Figure2B). We next examined the effect of orexin A on the transcription of Id-1, a target for BMP-receptor signaling. As shown in Figure2C, BMP-4 (10 ng/mL) treatment for 1 h significantly upregulated Id-1 mRNA, Int. J. Mol. Sci. 2021, 22, 4553 3 of 10

Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 3 of 10 and co-treatment with orexin A (100 nM) suppressed the expression of Id-1 mRNA induced by BMP-4.

Figure 1. Expression of orexin receptors and the effect of orexin A on CRH CRH receptor receptor and and POMC POMC expression expression in in AtT20 AtT20 cells. cells. (A) The expression expression of of mRNAs mRNAs encoding encoding OX1R, OX1R, OX2R OX2R,, and and RPL19 RPL19 was was examined examined by RT by− RTPCR−PCR in AtT20 in AtT20 cells cellscompared compared with withthat in that mouse in mouse gonadotrope gonadotrope LβT2 Lcells.βT2 MM: cells. molecular MM: molecular weight weight marker. marker. (B) After (B) preculture, After preculture, cells were cells treated were treated with orexin with (ORX)orexin in (ORX) serum in-free serum-free media. After media. 24− Afterh culture, 24-h total culture, cellular total RNA cellular was RNAextracted, was and extracted, after 24 and−h or after 48−h 24-h culture, or 48-h cellculture, lysates werecell lysates extracted. were The extracted. mRNA Theexpression mRNA levels expression of Crhr1 levels were of quantified Crhr1 were by quantified qPCR, and by the qPCR, expression and the levels expression of target levels of weretarget standardized genes were standardized by the RPL19 by the level RPL19 in each level sample. in each The sample. cell Thelysates cell were lysates analyzed were analyzed by immunoblotting by immunoblotting (IB) with (IB) anti−CRHR1 and anti-actin. (C) After preculture, cells were treated with ORX alone or with the combination of ORX and with anti−CRHR1 and anti-actin. (C) After preculture, cells were treated with ORX alone or with the combination of ORX CRH in serum-free media. After 24−h culture, total cellular RNA was extracted, and the mRNA expression levels of POMC and CRH in serum-free media. After 24-h culture, total cellular RNA was extracted, and the mRNA expression levels of were quantified by qPCR. The expression levels of target genes were standardized by the RPL19 level in each sample. ResultsPOMC wereare shown quantified as means by qPCR. ± SEM The of expressiondata from at levels least ofthree target independent genes were experiments. standardized Statistical by the RPL19 analysis level was in each performed sample. byResults the unpaired are shown t− astest means (B) or± ANOVASEM of data(B,C). from * p < at 0.05 least vs. three between independent the control experiments. groups. Statistical analysis was performed by the unpaired t−test (B) or ANOVA (B,C). * p < 0.05 vs. between the control groups. Next, we examined the effect of orexin on the action of BMP-4 in AtT-20 cells. We To elucidate the mechanism by which orexin A inhibits BMP-Smad signaling, we previously reported that BMP-4 suppressed POMC mRNA expression in AtT20 cells [20]. further examined the expression levels of BMP-receptor components using real-time PCR. As shown in Figure 2A, BMP-4 (10 ng/mL) treatment for 24 h decreased POMC mRNA, As shown in Figure3A, the expression levels of both BMP type I receptors (ALK2, ALK3, consistent with the results of our previous study. Co-treatment with orexin (10–100 nM) and ALK6) and type II receptors (BMPRIII and ACTRIIA) were unchanged after 24 h of did not change the POMC mRNA expression induced by BMP-4 treatment from 1 h to 24 treatment with orexin A (100 nM). Next, we examined the effect of orexin A on inhibitory h (Figure 2a). On the other hand, focusing on the effect of orexin on BMP-Smad signaling, Smads. Both Smad6 and Smad7 are known as inhibitory Smads, and they interfere with stimulation with BMP-4 (10 ng/mL) for 1 h activated Smad1/5/9 phosphorylation in AtT20 the receptor-regulated phosphorylation of other Smad molecules. Treatment with orexin A cells (Figure 2b). Importantly, orexin A (100 nM) pretreatment for 24 h inhibited the phos- (100 nM) for 24 h enhanced the mRNA expression of both inhibitory Smad6 and Smad7 phorylation of Smad1/5/9 by BMP-4 (Figure 2b). We next examined the effect of orexin A (Figure3B). To examine whether the changes in the inhibitory Smads can be detected at the on the transcription of Id-1, a target gene for BMP-receptor signaling. As shown in Figure protein level, Western blot analysis was also performed. As a result, treatment with orexin 2c, BMP-4 (10 ng/mL) treatment for 1 h significantly upregulated Id-1 mRNA, and co- A (100 nM) for 24 h significantly increased the protein expression levels of Smad6 and treatmentSmad7 (Figure with3 C).orexin These A (100 findings nM) suggested suppressed that the orexin expression A was involvedof Id-1 mRNA in the BMP-Smadinduced by BMPsignaling-4. in AtT20 cells through the upregulation of inhibitory Smad6/7.

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Figure 2. Effects of orexin A on BMP−receptor signaling in AtT20 cells. (A) After preculture, AtT20 cells were treated Figure 2. Effectswith of the orexin combination A on BMP of orexin−receptor (ORX) and signaling BMP−4 in serumAtT20−free cells. media. (A) After After 24preculture,−h stimulation AtT20 with cells orexin were and treated with the combination1− ,of 6− orexin, and 24− (ORX)h stimulations and BMP with− BMP4 in− serum4, total cellular−free media. RNA was After extracted, 24− andh stimulation the mRNA expression with orexin levels of and POMC 1−, 6−, and 24−h stimulations withwere BMP quantified−4, total by qPCR. cellular The expression RNA was levels extracted of target genes, and werethe standardizedmRNA expression by the RPL19 levels level of in eachPOMC sample. were quantified by qPCR. The (expressionB) Cells were levels pretreated of target with ORX genes in serum were−free standardized media for 24 h.by Afterthe RPL19 60-min stimulationlevel in each with sample. BMP−4, the (B) cell Cells were pre- lysates were subjected to immunoblot (IB) analysis using anti−pSmad1/5/9, anti−tSmad1, and anti−actin. (C) Cells treated with ORXwere pretreatedin serum with−free ORX media in serum for− free24 mediah. After for 2460 h.-min After stimulation 60−min stimulation with with BMP BMP−4,− 4,the total cell cellular lysates RNA were was subjected to immunoblot (IB)extracted, analysis and mRNA using levels anti of−pSmad1/5/9, Id−1 were examined anti by−tSmad1 qPCR. The, and expression anti− levelsactin of. target(C) Cells genes werewere standardized pretreated by with ORX in serum−free mediathe RPL19 for 24 level h. in After each sample.60−min Results stimulation are shown with as means BMP±SEM−4, total of data cellular from at least RNA three was independent extracted experiments., and mRNA levels of Id−1 were examinedStatistical by analysis qPCR. was The performed expression by ANOVA. levels Values of target with different genes superscript were standardized letters are significantly by the differentRPL19 at levelp < 0.05 in each sample. Results are shown(B,C). as * p means< 0.05 vs. ±SEM between of the data control from groups at least (A). three independent experiments. Statistical analysis was performed by ANOVA. Values with different superscript letters are significantly different at p < 0.05 (B,C). * p < 0.05 vs. between the control groups (A).

To elucidate the mechanism by which orexin A inhibits BMP-Smad signaling, we further examined the expression levels of BMP-receptor components using real-time PCR. As shown in Figure 3a, the expression levels of both BMP type I receptors (ALK2, ALK3, and ALK6) and type II receptors (BMPRIII and ACTRIIA) were unchanged after 24 h of treatment with orexin A (100 nM). Next, we examined the effect of orexin A on inhibitory Smads. Both Smad6 and Smad7 are known as inhibitory Smads, and they interfere with the receptor-regulated phosphorylation of other Smad molecules. Treatment with orexin A (100 nM) for 24 h enhanced the mRNA expression of both inhibitory Smad6 and Smad7 (Figure 3b). To examine whether the changes in the inhibitory Smads can be detected at the protein level, Western blot analysis was also performed. As a result, treatment with orexin A (100 nM) for 24 h significantly increased the protein expression levels of Smad6 and Smad7 (Figure 3c). These findings suggested that orexin A was involved in the BMP- Smad signaling in AtT20 cells through the upregulation of inhibitory Smad6/7.

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Figure 3. Effects of orexin A on the expression of BMP-receptor signaling molecules in AtT20 cells. (A,B) AtT20 cells treated with orexin (ORX) for 24 h were extracted, and the mRNA expression levels of Alk2, Alk3, Alk6, BmprII, ActrIIa (A), Smad6, Figure 3. Effectsand of Smad7 orexin (B) A were on quantifiedthe expression by qPCR. of The BMP expression-receptor levels signaling of target genes molecules were standardized in AtT20 by cells. the RPL19 (A,B level) AtT20 in cells treated with orexin (OReachX) sample. for 24 (C )h Cells were were extracted treated with, ORXand inthe serum mRNA−free media expression for 24 h. Thelevels cell lysates of Alk2, were subjectedAlk3, Alk6, to immunoblot BmprII, ActrIIa (A), Smad6, and Smad7(IB) analysis (B) were using antibodiesquantified that by detect qPCR. Smad6, The Smad7, expression and actin. levels Results of are target shown genes as means were±SEM standardized of data from at by the RPL19 level in each sample.least three ( independentC) Cells were experiments. treated Statistical with ORX analysis in was serum performed−free by media the unpaired for 24t-test. h. The * p < 0.05cell vs.lysates between were the subjected to immunoblot (IB)control analysis groups. using antibodies that detect Smad6, Smad7, and actin. Results are shown as means ±SEM of data from at least three independent experiments.3. Discussion Statistical analysis was performed by the unpaired t-test. * p < 0.05 vs. be- tween the control groups. In the present study, we investigated the functional roles of orexin in CRH and BMP signaling by AtT20 cells. As for the orexin receptors, OX2R rather than OX1R was predominantly expressed in AtT20 cells. Orexin A enhanced CRHR1 expression and 3. Discussionincreased POMC mRNA expression in the presence of CRH. Regarding the BMP receptor In signaling,the present orexin study, A treatment we investigated suppressed BMP-4-induced the functional Smad1/5/9 roles signalingof orexin and in Id- CRH and BMP 1 mRNA expression by AtT20 cells. It was also revealed that the BMP signaling was signalingmodulated by AtT20 by orexin cells. A through As for the the upregulation of inhibitorys, Smad6/7OX2R rather expression. than These OX1R was pre- dominantlyresults indicate expressed that orexin in AtT20 affects the cells. transcriptional Orexin A regulation enhanced of POMC CRHR1 through expression CRH- and in- creasedand POMC BMP-Smad mRNA signaling expression in corticotrope in the cells presence (Figure4). of CRH. Regarding the BMP receptor signaling, orexin A treatment suppressed BMP-4-induced Smad1/5/9 signaling and Id-1 mRNA expression by AtT20 cells. It was also revealed that the BMP signaling was modu- lated by orexin A through the upregulation of inhibitory Smad6/7 expression. These re- sults indicate that orexin affects the transcriptional regulation of POMC through CRH- and BMP-Smad signaling in corticotrope cells (Figure 4).

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Figure 4. Functional roles of orexin A in the activities of CRH and BMP−4 in corticotrope cells. OX2R rather than OX1R was predominantly expressed in corticotrope AtT20 cells. Orexin A up- regulated CRHR1 expression and CRH−induced POMC transcription. Orexin A also suppressed BMP−4−induced Smad1/5/9 phosphorylation and Id−1 transcription by upregulating inhibitory Smad6 and Smad7. Thus, orexin A has an enhancing effect on POMC expression by upregulating Figure 4. Functional roles ofCRH orexin signaling A in the and activities downregulating of CRH and BMP BMP−Smad−4 in action, corticotrope which is cells. possibly OX2R involved rather than in the OX1R control of was predominantly expressedstress in responsescorticotrope and AtT20 their resilience.cells. Orexin A upregulated CRHR1 expression and CRH−induced POMC transcription. Orexin A also suppressed BMP−4−induced Smad1/5/9 phosphorylation and Id−1 transcription by upregulating inhibitory Smad6 andIt has Smad7. been Thus, reported orexin that A has orexin an enhancing is closely effect related on POMC to the expression regulation by upregu of the HPAlating axis. CRH signaling and downregulatingOX1R is BMP widely−Smad distributed action, which in the is hypothalamus,possibly involved while in the OX2R,control butof stress not OX1R, responses is predomi- and their resilience. nantly distributed in the PVN, in which CRH and AVP neurons are located [23]. It has also been reported that OX2R antagonists attenuated the increase in ACTH induced by stress and orexinIt has stimulation,been reported indicating that orexin that is OX2R closely is functionally related to the involved regulation in the of stress-mediated the HPA axis. orexinOX1R is action widely of thedistributed HPA axis in activation the hypothalamus, in the hypothalamus while OX2R, [24 but,25 ].not In OX1R, the present is predom- study, OX2Rinantly was distributed found to in be the expressed PVN, in in which the mouse CRH pituitaryand AVP tumorneurons cell are line located AtT20 [23] as well. It has as inalso human been pituitaryreported that corticotrope OX2R antagonists cells, suggesting attenuated that OX2Rthe increase is involved in ACTH in the induced control by of ACTH,stress and not orexin only in stimulation, the hypothalamus indicating but that also OX2R in the is pituitary. functionally involved in the stress- mediatedOf interest, orexin inaction the presentof the HPA study, axis orexin activation A was in the found hypothalamus to enhance [24,25] the expression. In the pre- of CRHR1,sent study, leading OX2R to was an enhancementfound to be expressed of CRHsignaling in the mouse by AtT20 pituitary cells. tumor Theeffect cell li ofne orexinAtT20 onas well the expression as in human of CRHpituitary receptors corticotrope has also cells, been suggesting reported. that For instance,OX2R is involved the expression in the ofcontrol CRHR1 of ACTH was increased, not only byin repeatedthe hypothalamus stress in rats,but also and in the the receptor pituitary. upregulation was attenuatedOf interest, by the in administrationthe present study, of orexin orexin receptor A was found antagonists to enhance [26,27 the]. Thoseexpression studies of suggestedCRHR1, leading that stress-induced to an enhancement orexin of stimulation CRH signaling promotes by AtT20 CRHR1 cells. expression, The effect consistent of orexin withon the the expression results of of the CRH present receptors study. has Since also orexin been neuronsreported. are For also instance, expressed the expressio in CRHR1n andof CRHR1 have physiological was increased input by repeated from CRH stress [8], in there rats, is and a very the close receptor interaction upregulation mediating was stressattenuated response by the between administration orexin and of orexin CRH signaling. receptor antagonists [26,27]. Those studies sug- gestedRegarding that stress the-induced direct effect orexin of orexin stimulation on pituitary promotes corticotropes, CRHR1 expression, Samson and consis Taylortent reportedwith the results that orexin of the alone present had study. no effect Since on orexin ACTH neurons secretion are but also that, expressed in the presence in CRHR1 of CRH,and ha orexinve physiological A inhibited input CRH-induced from CRH ACTH [8], there secretion is a very more close potently interaction than orexin mediating B in ratstress pituitary response primary between cultured orexin cells and [CRH28]. Their signaling. findings are partially inconsistent with the resultsRegarding of the present the direct study, effec possiblyt of orexin due on to differencespituitary corticotropes, in the types Samson of cells used.and Taylor They suggestedreported that that orexin the effect alone of had orexin noon effect corticotropes on ACTH was secretion mediated but bythat the, in OX1R-mediated the presence of PKC pathway [28], whereas OX2R was predominantly detected by RT-PCR in AtT20 cells in the present study. It should be taken into account that the expression of orexin receptors in pituitary cells has been reported to be varied among animal species [29]. AtT20 cells have

been shown to have some differences from native corticotropes in endocrine activity [30], Int. J. Mol. Sci. 2021, 22, 4553 7 of 10

and those differences might be related to the difference between the results of the present and previous studies. We have been focusing on BMP signaling in various tissues [17]. BMP stimulation is converted into phosphorylation of Smad1, Smad5, and Smad9 (Smad1/5/9), which, to- gether with Smad4, called Co-Smad, translocate to the nucleus and regulate the expression of various genes. Inhibitory Smads, such as Smad6 and Smad7, suppress this phospho- rylation and negatively regulate the signal. We have also revealed an interrelationship between orexin and BMP signaling in various endocrine systems. In the pituitary, orexin A suppressed PRL production by inhibiting BMP-4/Smad signaling in rat somatolactotrope GH3 cells [22]. In the ovary, orexin A enhanced FSH-stimulated production by suppressing BMP-6/Smad signaling in granulosa cells [31]. The common link between those results and the results of the present study was the finding that orexin upregulated inhibitory Smad6/7. A functional link between orexin and inhibitory Smad has also been reported by other laboratories. Orexin A treatment affected the expression of a variety of genes related to cell proliferation and metabolism in HEK293 cells overexpressing orexin receptors [32], in which the BMP-Smad regulation with enhanced Smad7 expression was functionally involved. However, it is still uncertain what intracellular signals of orexin A can modulate the BMP-Smad signaling. We have also reported that , a circadian hormone, can regulate BMP-Smad signaling via inhibitory Smad6/7 [33]. Considering that BMP-4 and melatonin are mutually linked in the control of clock gene expression in AtT20 cells [34], this novel interaction between BMP and orexin signaling might also be involved in the regulation of the circadian profile of the HPA axis.

4. Materials and Methods 4.1. Reagents CRH, Dulbecco’s Modified Eagle’s Medium (DMEM), and DMEM high were purchased from Sigma-Aldrich Corp. (St. Louis, MO, USA). Recombinant human BMP-4 was purchased from R&D Systems Inc. (Minneapolis, MN, USA), and human orexin A was purchased from FUJIFILM Wako Pure Chemical Corp. (Osaka, Japan).

4.2. Cell Culture Mouse corticotrope AtT20/D16v (AtT20) cells under passage 20 were cultured in DMEM supplemented with 10% (v/v) fetal calf serum (FCS), penicillin and streptomycin in ◦ 12-well plates under a 5% (v/v) CO2 atmosphere at 37 C. Mouse gonadotrope LβT2 cells were cultured in DMEM high glucose supplemented with 10% (v/v) FCS and antibiotics. Af- ter preculture with the growth medium, the medium was changed to a serum-free medium.

4.3. RNA Extraction and Quantitative PCR After preculture, AtT20 cells (1 × 105 viable cells) were transferred to a serum-free medium and treated with orexin A alone, or with a combination of CRH and orexin A for 24 h. In experiments using co-treatment with BMP-4 and orexin A, the cells were transferred to a serum-free medium and treated with orexin A for 24 h, while the treatment time of BMP-4 was set between 1 h and 24 h. The culture medium was then removed, and total cellular RNA was extracted using TRI Reagent® (Cosmo Bio Co., Ltd., Tokyo, Japan). Primer pairs for PCR were selected from different exons of the corresponding genes as follows: mouse OX1R: 1261-1283 and 1660-1682 (BC119583.2); OX2R: 1216-1238 and 1422-1443 (BC140957.1); and BMP type II receptor (BMPRII): 1787-1806 and 1944-1963 (NM_ 007561.4). Mouse POMC, Id-1, Smad6, Smad7, ALK2, ALK3, ALK6, ACTRIIA, and ribosomal protein L19 (RPL19) were selected as we reported previously [21]. The extracted RNA (1 µg) was subjected to an RT reaction using ReverTra Ace® (TOYOBO CO., LTD., Osaka, Japan) with a random hexamer and deoxynucleotide triphosphate (dNTP). After optimizing the annealing conditions for each pair of primers, quantitative PCR was performed to quantify the level of target gene mRNA using the LightCycler 96 System (Roche Diagnostic Co., Tokyo, Japan). The relative expression of each mRNA was Int. J. Mol. Sci. 2021, 22, 4553 8 of 10

determined by the ∆∆Ct method, in which ∆Ct was the value obtained by subtracting the Ct value of RPL19 mRNA from that of the target mRNA. The amount of target mRNA relative to RPL19 mRNA was expressed as 2−(∆∆Ct), and the results were expressed as the ratio of target mRNA to RPL19 mRNA.

4.4. Western Immunoblotting Cells (1 × 105 viable cells) were precultured with DMEM containing 10% (v/v) FCS and antibiotics in 12-well plates. After preculture, the medium was changed to a serum-free medium and treated with orexin A for from 24 h to 48 h. In experiments using co-treatment with BMP-4 and orexin A, the cells were stimulated with the indicated concentrations of BMP-4 for 60 min after a 24 h treatment with orexin A. The treated cells were solubilized in 100 µL RIPA lysis buffer (Upstate Biotechnology, Lake Placid, NY, USA) containing 1 mM Na3VO4, 1 mM NaF, 2% (v/v) SDS, and 4% (v/v) β-mercaptoethanol. Western blot analysis was performed using the cell lysates with specific antibodies against phospho- Smad1/5/9 (pSmad1/5/9; Cell Signaling Technology, Inc., Beverly, MA, USA; catalog #13820), total-Smad1 (tSmad1; Cell Signaling Technology; catalog #6944), CRH receptor type-1 (CRHR1; Aviva Systems Biology, San Diego, CA, USA; catalog #OASG01837), Smad6 (Cell Signaling Technology; catalog #9519), Smad7 (R&D Systems; catalog #MAB2029) and actin (Sigma-Aldrich Co. Ltd., St. Louis, MO, USA; catalog #A2066). The blotted bands were analyzed by the C-DiGit® Blot Scanner System (LI-COR Biosciences, Lincoln, NE, USA) by scanning the integrated signal intensities. To evaluate the phosphorylated Smad contents, the ratios of the digitized levels of pSmad/tSmad bands were calculated. For CRHR1, Smad6, and Smad7 protein levels, the ratios of the digitized levels of each actin band were calculated.

4.5. Statistical Analysis Experimental results are shown as means ±SEM of data from at least three indepen- dent experiments, each performed with triplicate samples. The data were then subjected to ANOVA followed by the Tukey post hoc test or unpaired t-test. All statistical analyses were performed using EZR Ver 1.42 (Saitama Medical Center, Jichi Medical University, Saitama, Japan) and the interface for R Ver 4.0.0 (The R Foundation for Statistical Computing, Vienna, Austria). p values < 0.05 were accepted as statistically significant.

5. Conclusions Collectively, our results showed that orexin modulated POMC transcription by en- hancing the expression of CRH receptors as well as suppressing BMP-Smad signaling. Our results suggest that orexin plays a modulatory role in the secretion of ACTH from the anterior pituitary. From a clinical point of view, orexin receptor antagonists, such as and , currently used in the treatment of , have been re- ported to decrease ACTH in rats and humans [35,36]. Control of orexin signaling might be a new strategy for stress management and/or treatment of Cushing’s disease by inhibiting POMC transcription in corticotropes.

Author Contributions: F.O. conceived and designed the experiments; S.F., M.K. and N.T.-Y. per- formed the experiments; S.F. analyzed the data; N.I. and T.N. contributed reagents/materials/analysis tools; and S.F., J.W. and F.O. wrote the paper. All authors have read and agreed to the published version of the manuscript. Funding: The present work was supported by Grants-in-Aid for Scientific Research (No.18K08479, 19K17985 and 21K08556) and Ofuji Endocrine Medical Award. Institutional Review Board Statement: Ethical review and approval were waived for this study, due to not applicable for studies not involving humans or animals. Informed Consent Statement: Not applicable for studies not involving humans. Data Availability Statement: Data is contained within the article. Int. J. Mol. Sci. 2021, 22, 4553 9 of 10

Conflicts of Interest: The authors declare no conflict of interest.

Abbreviations

ACTH adrenocorticotropin ActRII activin type II receptor ALK activin receptor-like kinase AVP arginine vasopressin BMP bone morphogenetic protein BMPRII BMP type II receptor CRH corticotropin-releasing hormone CRHR CRH receptor HPA hypothalamic-pituitary-adrenal ORX orexin A OX1R orexin type 1 receptor OX2R orexin type 2 receptor POMC pro-opiomelanocortin PVN paraventricular nucleus (TGF)β transforming growth factor (TGF)β

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